How ppGpp, pppGpp works: specific structural and regulatory interactions with RNA polymerase. This year we have achieved a significantly more rigorous definition of the specificity of interactions between ppGpp, pppGpp and the RNA polymerase protein (see Mechold et al., 2013). This achievement required meeting four challenges. Two of these were achieved in our lab over the past decade and other two over the last two years. 1. Stressed cells normally accumulate various mixtures of the two regulators so we devised conditions to gratuitously induce cellular accumulation of either one or the other nucleotide. If regulatory activities are shared but not unique then estimates of relative regulatory activities can be assigned to each in vivo. Ideally, the induction condition must occur during balanced growth and cannot itself have physiological consequences (ie constitute a stress) other than those due to (p)ppGpp. 2. Quantitative cellular assays are needed to allow quantitative comparisons of (p)ppGpp regulation. 3. Conditions are needed that allow synthesis of pure preparations of ppGpp as well as pppGpp for use as reagents to assess in vitro biochemical regulatory activities of each nucleotide. 4. Conditions and reagents are needed that allow documentation of regulation in pure in vitro transcription reactions to dissect mechanisms in order to rule out indirect effects prevalent for in vivo regulation. The reagents are pure RNA polymerase holoenzyme (core + sigma70), pure DksA protein and promoter templates that display positive or negative (p)ppGpp regulation in vivo and in vitro. 5. Structural studies of the ppGpp or pppGpp binding sites to the RNA polymerase require soaking crystals of RNA polymerase protein with each nucleotide. This crucial challenge was met in collaboration with Dr. K.S. Murakami, who only this year succeeded in obtaining the crystal structure of E. coli RNA polymerase holoenzyme. Previous claims of RNA polymerase binding sites that were subsequently proven spurious employed proteins either from thermophiles or E. coli core enzyme, which lacks the sigma subunit critical for the initiation step of transcription regulated by (p)ppGpp. ppGpp is a more potent than pppGpp as regulator of growth rate in vivo and of rRNA transcription initiation in vitro. Cellular induction of ppGpp alone was found to be 10-fold more potent an inhibitor of balanced growth than induced pppGpp; this effect on growth rate itself is independently observed to interlock with consensus estimates of observed changes accompanying induction of ratios of RNA/DNA. The gold standard of growth rate control by (p)ppGpp is a function of RNA/DNA ratios. Moreover, we are confident that effects of induction are due to the (p)ppGpp nucleotides and not to other growth toxic effects accompanying induction because neither growth inhibition nor RNA/DNA ratio changes occur when missense mutants of either (p)ppGpp synthetase proteins are induced. Transcription reactions were observed to confirm this observation of regulator specificity. There is a consensus that (p)ppGpp regulation of growth primarily reflects inhibition of total rRNA synthesis measured in vitro as rrnB P1 promoter transcription initiation activity. Systematic in vitro measurements of effects of pure ppGpp and pppGpp on transcription initiation documented that ppGpp is also a much more potent inhibitor than pppGpp. This comprises the first observation of pppGpp regulatory activity on transcription in vitro. (p)ppGpp regulation activation: ppGpp is a more potent regulator in vitro and in vivo. A well documented example of promoter activation by (p)ppGpp in vivo is regulation of the threonine operon transcription. We therefore compared effects of ppGpp and pppGpp on in vitro transcription initiation by the threonine operon promoter (pthr). The regulatory activity of ppGpp was found to be a dramatically more potent activator than pppGpp. The comparison reveals the potency for regulation is similar as for the rrnB P1 promoter but the regulatory effect is activation rather than inhibition. We also studied an example where activation by (p)ppGpp is much more complex than simple promoter activation. This is regulation of the alternative sigma factor RpoS, which governs stationary phase gene expression. Here, regulation involves multiple components that include post-transcriptional elements. These studies provided the fifth example of an emerging pattern: ppGpp is a more potent regulator than pppGpp. Regulation by one analog was always shared with the other; no examples were found of regulation unique to one and not the other. A single (p)ppGpp binding site exists at the interface between the omega and beta-prime subunits of RNA polymerase holoenzyme. A single site for both nucleotides was found by Dr. Murakami when either 1 mM ppGpp or pppGpp was added to existing crystals of RNA polymerase and resolved at 3.9 or 4.2 Angstroms, respectively. The site is located at the interface between the amino terminus of the omega (rpoZ) and beta- (rpoC) subunits. The site is 30 Angstroms distant from the catalytic site with no evidence of a site near the catalytic center. Interestingly, the amino terminal methionine of omega is not visualized in the crystal structure and was found by mass spectrometry to be cleaved post-translationally. If this residue was present it would partially obstruct the binding. A few weeks after our paper appeared (Apr 25), two other groups labs of Tom Steitz- Yuo et al. Mol Cell. 2013 May 9;50(3):430-6 and Rick Gourse- Ross et al. Mol Cell. 2013 May 9;50(3):420-9 reported a similar binding site for ppGpp that differs from ours only with respect to the orientation of ppGpp; pppGpp was not studied. Comments on all three papers may be found in: Kharlstrom CT, Nat Rev Microbiol. 2013 Jul; 11(7):429. Does the (p)ppGpp binding site function in regulation? This question is not yet resolved in our view. The Ross paper presents evidence that point mutants in omega and beta-prime subunits predicted to be key residues from the site structure do alter measured binding affinities of ppGpp for polymerase and improve promoter open complex stabilities that are normally destabilized by ppGpp and DksA. However in vivo evidence of altered regulation due to single point mutants in either omega or beta-prime remains nonexistent. Resistance, defined as the absence of expected regulatory effects when (p)ppGpp is verifiably present has not yet been demonstrated. The question of functionality of this site is of keen interest for us. This particularly applies with respect to implication of the sigma subunit because over two decades ago we constructed the first omega deletion mutant and reported that the deficiency did not perturb normal (p)ppGpp regulation during amino acid starvation (Gentry et al J. Bacteriol. 1991, 173(12):3901). We recently have validated our earlier conclusions with ppGpp-sensitive growth tests that two different omega deletion alleles do not alter the (p)ppGpp regulatory responses.